1. Field of the Invention
This invention relates to the preservation and restoration of hemoglobin as an oxygen transport vehicle in blood substitutes. More particularly, this invention relates to compositions and methods of restoring hemoglobin which has been converted to methemoglobin.
2. Description of the Prior Art
In many instances, oxygen is a toxic or otherwise destructive agent. A wide variety of compounds have been studied and used to prevent oxidative damage in biological systems. Metal ions such as the ferric ion are often catalysts in the production of oxidizing species in solution. Desferal, which is a specific chelator of this form of iron, has been used successfully to block the oxidative catalyst action of the ferric ion. Other chelators such as EDTA and ascorbate have been used to remove metal ion catalysts. Ascorbate is a frequently used antioxidant although it is known to produce H.sub.2 O.sub.2 by reacting with oxyhemoglobin. Prooxidant effects have been reported at 0.5 mM ascorbate and antioxidant effects at 5 mM concentrations.
The conversion of H.sub.2 O.sub.2 to hydroxyl and superoxide radicals usually results in oxidative damage to proteins and other important molecules in cells and their metabolic removal is an important step in protection of cells. Radical scavengers such as the carbohydrates glucose and mannitol have been shown by others to be effective in the removal of deleterious radicals.
Much work has been done over many years to develop a human blood substitute that will carry oxygen. The three approaches which have emerged as leaders in this endeavor are; modified hemoglobin, synthetic organic materials such as fluorocarbons and artificial red blood cells (RBC). Of these approaches, artificial red blood cells most closely approximate the normal physiological system.
One form of artificial red blood cell is formed by encapsulating a hemoglobin solution as the aqueous phase in a lipid membrane as the oil phase. This lipid encapsulated hemoglobin system is known as LEH. Some methods of preparation and use for these functional surrogate red blood cells are described by T. M. S. Chang, "Semipermeable Microcapsules", Science, 146, 524, (1964); L. Djordjevich, and I. F. Miller, "Synthetic Erythrocytes from Lipid Encapsulated Hemoglobin", J. Exp. Hemat. 8, 584, (1980); and C. A. Hunt, R. L. Burnette, R. D. MacGregor, A. E. Strubbe, D. T. Lau, N. Taylor and H. Kawada, "Synthesis and Evaluation of a Prototypal Artificial Red Cell", Science, 230, 1165, (1985).
One Of the major barriers to the successful use of artificial red blood cells and other hemoglobin (Hb) based blood substitutes is a means for preserving the preparations against the oxidation of Hb during preparation and storage. The oxidized form of Hb, methemoglobin (metHb), will not transport oxygen, and the accumulation of metHb often results in the precipitation of protein in the form of Heinz bodies, Bunn, Semin. Hematol., 9:3, (1972). Oxidative damage is also the principle limiting factor in long-term storage of Hb based blood substitutes including LEH.
The above cited studies do not address the question of methemoglobin formation and make no attempt to prevent or reverse its formation from hemoglobin. J. Szebeni, J. H. Breuer, J. G. Szelenyl, G. Bathori, G. Lelkes and S. R. Hollan, in "Oxidation and Denaturation of Hemoglobin Encapsulated in Liposomes", Biochim. Biophys. Acta, 798, 60, 1984, recognize that oxidation of hemoglobin to nonfunctional methemoglobin is a problem that must be solved in order to produce a functional artificial RBC.
Previous work has indicated that much of the oxidative damage to hemoglobin is induced via H.sub.2 O.sub.2 generation and superoxide radicals. Chiu et al., Free Radicals in Biology, Vol. 5, pp.115-160, ed. L. Parker, Academic Press, (1982). Thus, such agents as catalase, superoxide dismutase (SOD), ascorbate, nicotinamide adenine dinucleotide/nicotine adenine dinucleotide phosphate (NADH/NADPH), and 3-ribosyluric acid have shown moderate protection of hemoglobin from oxidative damage from nitrites and radical producing agents. It is known that the oxidation of hemoglobin to methemoglobin is reversible in vivo, but this reversal has not been effected in an artificial RBC.
J. Szebeni, C. C. Winterbourn and R. W. Carrell, in "Oxidative Interactions Between Haemoglobin and Membrane Lipid, A Liposome Model", Biochem. J., 220, 685, 1984, teach that oxidation of the lipid in the membrane and oxidation of the encapsulated hemoglobin are interrelated. The writers suggest that certain ingredients such as catalase and 5 mM glutathione (GSH) may help prevent this oxidation when using fresh RBC lysate to make the liposomes. They reported no effect by GSH when using purified hemoglobin although they noted that catalase was beneficial. They did not report studies showing that any of these additives were able to reduce methemoglobin levels. All of their studies were carried out at 37.degree. C., which is not a favorable storage condition.
R. C. Smith and V. Nunn, in "Prevention by 3-N-Ribosyluric Acid of the Oxidation of Bovine Hemoglobin by Sodium Nitrite", Arch. Biochem. Biophys., 232, 348, 1984, teach that 3-N-ribosyluric acid, ascorbic acid and 0.1 mM GSH prevented oxidation of hemoglobin by nitrite at 37.degree. C. This oxidation protection was probably caused by interfering with the action of H.sub.2 O.sub.2. They did not report any studies at 4.degree. C., but at 37.degree. C. none of the antioxidants they studied reversed the oxidation of methemoglobin and, even in the presence of the antioxidants, all of the hemoglobin was converted to methemoglobin within 15 hours.
Sehgal et al., "Control of Methemoglobin Formation in Stroma-Free Hemoglobin Solutions", J. Surgical Research, 31, pp.13-17, (1981), disclose that NADH and NADPH will reduce methemoglobin formation in stroma-free hemoglobin preparations used in total exchange transfusion studies in baboons. All work was done in vivo and no evaluation of methemoglobin formation in preparations stored at 4.degree. C. was performed. Additional antioxidant agents such as ascorbate and glucose were also added to these preparations.
L. Djordjevich, J. Mayoral, I. F. Miller and A. D. Ivankovich, "Cardiorespiratory Effects of Exchange Transfusions with Synthetic Erythrocytes in Rats", Critical Care Medicine, 15, 318, 1987, employed low levels of glucose, L-ascorbic acid and GSH (between 0.3 and 1 mM) as substrates for methemoglobin-reducing enzymes. They did not report any effect of this in vivo treatment.
In both U.S. Pat. Nos. 4,425,334 and 4,612,370, Hunt suggests including an antioxidant in LEH preparations. Specifically, Hunt describes LEH preparations in which an antioxidant in the form of alpha-tocopherol is included in the oil or lipid phase of the LEH. Hunt does not suggest the incorporation of an antioxidant into the aqueous or Hb phase of the LEH. The production and stability of an effective LEH system will require protection from oxidative damage for both the lipid portion and the aqueous compartment of LEH.